Morphogenesis remodels the shape of the embryo, generating the myriad of complex forms and structures that characterize the mature organism. Defects in morphogenesis give rise to birth defects such as spina bifida and anencephaly. While the outcome of such morphogenetic defects is very apparent, the underlying cellular and molecular mechanisms of normal morphogenesis remain unclear. Investigating and characterizing morphogenesis is an important step understanding how to overcome birth defects. The studies proposed here focus on key a cellular aspect of morphogenesis, the generation of cell shape changes; signal transduction via the small GTPase molecular switch, RhoA, is critical for these cell shape changes. This proposal will extend previous studies, directly investigating the role of RhoA signal transduction during morphogenesis of Drosophila melanogaster. This allows exploitation of the powerful molecular and classical genetic techniques afforded by the model organism. This research will examine the precise nature of the defects in RhoA and RhoGEF2 mutant animals. This will be accomplished by utilizing fixed preparations of mutant embryos that have been stained with antibodies directed against the cytoskeleton. New mutant alleles of RhoA and its positive regulator, Rho Guanine Exchange Factor 2, will be generated in order to further define the morphogenetic processes that require Rho signal transduction. This will include standard chemical mutagenesis techniques and the generation of a conditional RhoA mutation. The relationship between Rho signal transduction and Myosin activation will be explored. This will be accomplished by molecularly engineering a myosin transgene and then expressing it in RhoA mutant embryos. Finally, powerful genetic strategies will be used to identify molecules that function upstream and downstream of Rho signal transduction. Tissue culture and biochemical studies have suggested a number of players in this process, but their biological relevance in the intact organism remains unclear. We will test these candidate loci directly in genetic interaction tests with RhoA mutations. Further, we will perform mutagenesis screens to identify novel gene products that collaborate in the Rho signal transduction pathway. Together, these studies will extend understanding of the fundamental role and pathways Rho signal transduction plays in morphogenesis.